A dozen or so monoclonal antibodies have been approved by the Food and Drug Administration (FDA) as human therapeutics including Orthoclone OKT3 for allograft rejection, ReoPro (abciximab) for adjunct treatment of percutaneous coronary intervention (PCI) including balloon angioplasty, atherectomy and stent placement, Rituxan for Non-Hodgkin's lymphoma, Simulet and Zenapax for organ rejection prophylaxis, Remicade for Rheumatoid arthritis and Crohn's disease, Synagis for respiratory syncytial virus (RSV), Herceptin for metastatic breast cancer, Mylotarg for acute myeloid leukemia and Campath for chronic lymphocytic leukemia, etc. These therapeutic antibodies can be divided into three major categories: murine monoclonal antibodies (Orthoclone OKT3); chimeric monoclonal antibodies (ReoPro, Rituxan, Simulet, and Remicade); and CDR-grafted monoclonal antibodies (Zenapax, Synagis, Herceptin, Mylotarg, and Campath). A murine monoclonal antibody is a mouse antibody; a chimeric antibody contains antibody of two or more species of animal, such as human and mouse; while CDR-grafted antibodies have lower amounts of foreign protein, generally in the complementarity determining region (CDR), thus the framework is human and the CDR are of mouse origin. In the above clinically approved antibodies, the non-human portion of the antibody derived is from a mouse antibody.
The mouse portion of the murine, chimeric or even CDR-grafted antibodies would elicit an immune response and associated side effects when administrated to a human, such as HAMA (human anti-mouse antibody) or HACA (human anti-chimeric antibody) responses. Thus, therapeutic antibody development is best suited with totally or 100% human antibodies.
There are two approaches in making human antibodies. One approach uses a human-mouse system such as the XenoMouse technology of Abgenix (Fremont, Calif.) or the HuAbMouse technology of Medarex, Inc. (Princeton, N.J.), wherein the host mouse immunoglobulin genes are inactivated and most of the human immunoglobulin genes are incorporated into the mouse to produce totally human antibodies in response to antigen stimuli in the mouse. Some of the difficulties in producing monoclonal antibodies with the human-mouse methodology include genetic instability, smaller antigenic specificities due to tolerance restriction of certain antibodies in a live animal, low throughput in access and screening of the in vivo antibody repertoire which can only be accessed via immunization with a selection on the basis of binding affinity and low production capacity.
The other approach is to generate libraries of antibody genes by cloning. Often the target genes are amplified prior to cloning.
There are a number of methods in the field of amplifying specific target nucleic acid sequences of interest. The polymerase chain reaction method (PCR), as described by Mullis et al., (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; and European Patent Application Nos. 86302298.4, 86302299.2, and 87300203.4, and Methods in Enzymology, Volume 155, 1987, pp. 335-350), is one of the most prominent methods. PCR involves the use of a pair of specific oligonucleotides as primers for the two complementary strands of the double-stranded DNA containing the target sequence. The primers are chosen to hybridize at the ends of each of the complementary target strands, 3′ of the target sequence. Template-dependent DNA synthesis, on each strand, can then be catalyzed using a thermostable DNA polymerase in the presence of the appropriate reagents. A thermal cycling process is required to form specific hybrids prior to synthesis and then to denature the double stranded nucleic acid formed by synthesis. Repeating the cycling process geometrically amplifies the target sequence.
A PCR method employing a reverse transcription step is also used with an RNA target using RNA-dependent DNA polymerase to create a DNA template. The PCR method has been coupled to RNA transcription by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR method, using the double-stranded DNA as a template for the transcription of single-stranded RNA. (see, e.g., Murakawa et al., DNA 7:287-295 (1988)). The PCR method has been applied to the amplification and cloning of the variable domain sequences of immunoglobulin or antibody genes (U.S. Pat. No. 6,291,158 to Winter et al. and U.S. Pat. No. 6,291,161 to Lerner et al.).
There are, however, several non-PCR-based amplification methods that can be used for amplifying specific target genes. One types of the non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets (see, e.g., Burg et al., WO 89/01050; Gingeras et al., WO 88/10315; Kacian and Fultz, EPO Application No. 89313154; Davey and Malek, EPO Application No. 88113948.9; Malek et al., WO91/02818 and U.S. Pat. No. 5,130,238; Davey et al., U.S. Pat. Nos. 5,409,818; 5,466,586; 5,554,517 and 6,063,603; Eberwine et al., U.S. Pat. No. 5,514,545; Lin et al., U.S. Pat. No. 6,197,554; and Kacian et al., U.S. Pat. No. 5,888,779).
Another type of amplification method uses a ligase chain reaction (LCR) as described in European Patent Publication No. 320,308. This method requires at least four separate oligonucleotides, two of which hybridize to the same nucleic acid template so their respective 3′ and 5′ ends are juxtaposed for ligation. The hybridized oligonucleotides are then ligated forming a complementary strand on the nucleic acid template. The double-stranded nucleic acid is then denatured, and the third and fourth oligonucleotides are hybridized with the first and second oligonucleotides that were joined together. The third and fourth oligonucleotides are then ligated together. Amplification is achieved by further cycles of hybridization, ligation, and denaturation.
Another amplification method uses Qβ replicase (Qβ) method as described in PCT Publication Ser. No. 87/06270 and U.S. Pat. No. 4,786,600 that uses a specific RNA probe which is capable of specific transcription by a replicase enzyme. The method requires the design and synthesis of RNA probes with replicase initiation sites.
Another type of amplification uses palindromic probes as described in EPO Publication Nos. 0427073A and 0427074A. The palindromic probe forms a hairpin with a nucleic acid target sequence. The probe contains a functional promoter located in the hairpin region from which RNA transcripts are produced.
There are also several versions of a strand displacement amplification method that uses one strand of DNA to displace same strand DNA sequences hybridized to their complementary DNA sequences to generate many copies of the target DNA sequences under isothermal conditions.
Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392-396 (January 1992), Walker et al., Nucl. Acids Res. 20:1691-1696 (1992), European Patent Application No. EP 0 497272, and European Patent Application No. EP 0 500 224, describe an oligonucleotide-driven amplification method using a restriction endonuclease. The restriction endonuclease nicks the DNA/DNA complex to enable an extension reaction and, therefore, amplification.
Becker et al., EPO Application No. 88306717.5, describe an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex cleaved prior to the extension reaction and amplification.
Dattagupta et al. described another version of the strand displacement amplification method by using a nucleic acid polymerase lacking 5′ exonuclease activity and a set of oligonucleotide primers to carry out isothermal amplification without requiring exonuclease activity or restriction endonuclease activity (U.S. Pat. No. 6,214,587).
Another amplification method is rolling circle amplification. The method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermal reaction that replicates the vector sequences exponentially (U.S. Pat. No. 6,287,824 to Lizardi).
A related amplification method using a similar approach is termed ramification extension amplification (RAM), U.S. Pat. No. 5,942,391 to Zhang et al. The RAM method involves hybridizing a target nucleic acid to several non-overlapping oligonucleotide probes that hybridize to adjacent regions in the target nucleic acid, the probes being referred to as capture/amplification probes and amplification probes, respectively, in the presence of paramagnetic beads coated with a ligand binding moiety. Through the binding of a ligand attached to one end of the capture/amplification probe and the specific hybridization of portions of the probes to adjacent sequences in the target nucleic acid, a complex comprising the target nucleic acid, the probes and the paramagnetic beads is formed. The probes may then ligate together to form a contiguous ligated amplification sequence bound to the beads, which complex may be denatured to remove the target nucleic acid and unligated probes.
Attempts to clone variable domain sequences of the immunological genes into an antibody framework vector and expressing the antibodies in a host cell such as in a phage using PCR have been described (U.S. Pat. No. 6,291,158 to Winter, et al.; and U.S. Pat. No. 6,291,161 to Lerner, et al.). Some of the difficulties in employing that PCR amplification scheme are that PCR amplification efficiency is dependent on both the primer and the template sequences. Certain sequences are preferentially amplified with other sequences being under-amplified or not amplified leading to under representation of the diversity of the resulting antibody libraries. An example of the limitations encountered when using PCR to clone a library is provided in Gao et al., Proc. Natl. Acad. Sci. (1999) 96:6025-6030.
The size of the human antibody repertoire is estimated to be on the order of 106 to 108 different antigen specificities. Exceptional larger numbers of specificities of the human antibodies can be generated by in vitro construction of VH and/or VL libraries by random recombination and shuffling, and saturation mutagenesis of the VH and VL DNA homologs.
One of the potential benefits of constructing human antibody libraries is to obviate the need for immunization by the generation of highly diverse “generic” human antibody libraries. In certain cases, very specialized human antibody libraries such as human antibody libraries made by using blood cells of cancer patients or blood cells of patients with autoimmune diseases such as rheumatoid arthritis, psoriasis, etc. may contain human antibodies with very high avidity and specificity for that particular diseases. Another benefit of having human antibody libraries is that they permit iterative cycles of mutagenesis or random recombination of the VH and VL gene repertoire to further optimize the specificity, affinity or catalytic properties of the immunoglobulins or their derivative antibodies such as Fab and scFv fragments.